Recombinant Danio rerio Prostaglandin E synthase 2 (ptges2)

What is Danio rerio Prostaglandin E Synthase 2 (ptges2) and what is its biological function?

Danio rerio Prostaglandin E Synthase 2 (ptges2), also known as microsomal prostaglandin E synthase 2 (mPGES-2), belongs to a family of three structurally and biologically distinct prostaglandin E2 synthases that catalyze the conversion of prostaglandin H2 (PGH2) to prostaglandin E2 (PGE2) . This enzyme is part of an important paracrine signaling system involved in numerous biological processes. In mammals, PGE2 plays crucial roles in the nervous system, including fever generation, sickness behavior, and nociception . The zebrafish ortholog likely participates in similar physiological processes, though species-specific differences may exist.

Unlike its counterpart mPGES-1, which is inducible and typically coupled with COX-2, mPGES-2 has been traditionally viewed as having a more constitutive role, though this view has been challenged by some studies showing regulated expression under certain conditions . Interestingly, knockout studies in mice have shown that mPGES-2 deletion produces no specific phenotype and no significant alteration in tissue levels of PGE2, raising questions about its precise physiological role .

What are the optimal storage and handling conditions for recombinant Danio rerio ptges2?

For optimal maintenance of protein activity and stability, recombinant Danio rerio ptges2 should be stored according to the following recommendations:

• Storage temperature: -20°C to -80°C for long-term storage

• Physical form: Typically provided as a lyophilized powder

• Reconstitution: The protein should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Stabilization: Addition of 5-50% glycerol (final concentration) is recommended for aliquots intended for long-term storage

• Working conditions: For short-term use, working aliquots can be stored at 4°C for up to one week

• Freeze-thaw cycles: Repeated freezing and thawing should be avoided as this can lead to protein denaturation and activity loss

To maintain maximum activity, proper aliquoting is necessary for multiple use. When first receiving the lyophilized powder, it is advisable to briefly centrifuge the vial prior to opening to bring all contents to the bottom .

How does recombinant Danio rerio ptges2 compare to mammalian orthologs?

While the search results don't provide direct sequence comparison data, we can draw several inferences about the relationship between Danio rerio ptges2 and its mammalian counterparts:

• Functional conservation: Like mammalian mPGES-2, Danio rerio ptges2 likely catalyzes the conversion of PGH2 to PGE2 as part of the arachidonic acid pathway

• Structural features: Both fish and mammalian versions appear to share key structural elements, including a thioredoxin-like fold that is characteristic of mPGES-2 enzymes

• Regulation: In mammals, mPGES-2 has been traditionally viewed as constitutively expressed, though some studies suggest regulated expression under certain conditions. Similar regulatory mechanisms may exist for Danio rerio ptges2

• Knockout studies: Studies in mice have shown that mPGES-2 deletion produces no specific phenotype and no significant alteration in tissue levels of PGE2. This raises questions about its precise physiological role across species

The availability of recombinant proteins from different species (mouse, cynomolgus, human, and zebrafish) suggests that comparative studies are possible and potentially informative for understanding the evolution and functional conservation of this enzyme family.

What are the best approaches for assessing Danio rerio ptges2 enzymatic activity?

Assessing the enzymatic activity of Danio rerio ptges2 requires careful experimental design considering the following methodology:

• Substrate preparation: PGH2 is unstable with a half-life of approximately 5 minutes at 37°C. Therefore, it should be stored in dry ice/acetone and handled in cold solvents. Working solutions should be prepared immediately before use.

• Reaction conditions: Based on studies of mammalian mPGES-2:

  • Buffer: Typically 100 mM Tris-HCl (pH 8.0)

  • Temperature: 22-25°C (room temperature)

  • Reaction time: 30-60 seconds due to PGH2 instability

  • Substrate concentration: 10-50 μM PGH2

  • Enzyme concentration: 0.5-5 μg of purified protein

• Activity measurement methods:

  • HPLC analysis of PGE2 formation

  • Enzyme immunoassay (EIA) for PGE2 quantification

  • Coupled spectrophotometric assay monitoring glutathione oxidation (if the zebrafish enzyme uses glutathione as a cofactor like some mammalian PGESs)

• Controls:

  • Heat-inactivated enzyme (negative control)

  • Commercial PGE2 standards

  • Known inhibitors of PGES activity as reference compounds

The enzymatic activity can be expressed as the amount of PGE2 produced per minute per milligram of protein under the specified conditions. Due to the lack of specific information on zebrafish ptges2 activity assays, optimization may be required when adapting protocols developed for mammalian orthologs.

How can I design and validate CRISPR/Cas9-based knockout models for ptges2 in zebrafish?

Designing and validating CRISPR/Cas9-based knockout models for ptges2 in zebrafish involves several critical steps:

• Guide RNA (gRNA) design:

  • Target early exons or functionally critical domains to ensure loss of function

  • Use zebrafish genome databases to identify unique target sequences in the ptges2 gene

  • Design 2-3 gRNAs targeting different regions to increase knockout efficiency

  • Check for potential off-target effects using tools like CHOPCHOP or CRISPOR

• CRISPR injection protocol:

  • Prepare Cas9 protein (or mRNA) and gRNAs

  • Inject into one-cell stage zebrafish embryos

  • Typical concentrations: 300 ng/μL Cas9 protein and 50-100 ng/μL gRNA

• Validation of mutations:

  • Primary screening: PCR amplification of the target region followed by T7E1 assay or heteroduplex mobility assay

  • Secondary confirmation: Direct sequencing of PCR products from individual F0 embryos

  • F0 mosaic adults should be outcrossed to generate F1 carriers with specific mutations

• Validation of knockout efficiency:

  • mRNA expression: qRT-PCR to assess ptges2 transcript levels

  • Protein expression: Western blot using anti-ptges2 antibodies

  • Enzymatic activity: Measure PGE2 production in tissue homogenates

• Phenotypic analysis:

  • Given the findings from mouse models showing no obvious phenotype , careful examination of:

    • Inflammatory responses

    • Neural development

    • Response to stimuli known to induce prostaglandin synthesis

    • Compensatory changes in other PGES family members

The establishment of zebrafish ptges2 knockouts would provide valuable insights, especially if the phenotype differs from the mammalian models, potentially revealing species-specific functions.

What approaches can be used to optimize expression and purification of active Danio rerio ptges2?

Optimizing expression and purification of active Danio rerio ptges2 requires addressing several technical challenges:

• Expression system selection and optimization:

  • E. coli is the established system for this protein , but consider these variables:

    • Strain selection: BL21(DE3), Rosetta, or SHuffle for proteins with disulfide bonds

    • Expression temperature: Lower temperatures (16-20°C) often improve folding

    • Induction conditions: IPTG concentration (0.1-1.0 mM) and duration (3-24 hours)

    • Media formulation: Rich media (TB, 2XYT) or minimal media with supplements

• Solubility enhancement strategies:

  • Fusion partners: In addition to His-tag, consider MBP, GST, or SUMO fusions

  • Chaperone co-expression: GroEL/ES, DnaK/J, or Trigger Factor

  • Lysis buffer optimization: Include mild detergents (0.1% Triton X-100, 0.5% CHAPS) if membrane-associated

• Purification optimization:

  • IMAC (Immobilized Metal Affinity Chromatography):

    • Ni-NTA or TALON resins with gradient elution

    • Buffer composition: pH 7.5-8.0, 300-500 mM NaCl, 5-10% glycerol

  • Secondary purification: Size exclusion chromatography or ion exchange

  • Tag removal: Consider TEV or PreScission protease cleavage if the tag affects activity

• Activity preservation:

  • Stabilizing additives: Glycerol (10-20%), reducing agents (DTT or β-ME)

  • Storage conditions: Flash freeze in liquid nitrogen and store at -80°C

  • Avoid repeated freeze-thaw cycles

• Quality control assessments:

  • SDS-PAGE and Western blot for purity and identity

  • Dynamic light scattering for aggregation state

  • Circular dichroism for secondary structure analysis

  • Activity assays as described in section 2.1

By systematically optimizing these parameters, researchers can improve the yield and quality of active recombinant Danio rerio ptges2.

How can structural studies of Danio rerio ptges2 contribute to inhibitor design?

Structural studies of Danio rerio ptges2 can significantly advance inhibitor design through several approaches:

• Structural determination methods:

  • X-ray crystallography: Requires high-purity, homogeneous protein samples

    • Crystallization screening with commercial kits

    • Co-crystallization with substrates, product, or known inhibitors

  • Cryo-EM: Emerging alternative for membrane-associated proteins

  • NMR: For dynamic regions or smaller domains of the protein

• Comparative modeling approaches:

  • Homology modeling using mammalian mPGES-2 structures as templates

  • Refinement based on molecular dynamics simulations

  • Validation through mutagenesis of predicted key residues

• Structure-based inhibitor design:

  • Active site mapping to identify key residues for catalysis

  • Virtual screening of compound libraries against the catalytic pocket

  • Fragment-based approaches to identify initial binding scaffolds

  • Structure-activity relationship studies to optimize lead compounds

• Selectivity considerations:

  • Comparative analysis with human PTGES2 to identify species-specific differences

  • Assessment of selectivity against other PGES family members

  • Molecular docking studies with potential inhibitors across multiple targets

• Validation experiments:

  • Thermal shift assays to confirm binding

  • Enzyme inhibition assays with purified protein

  • Cellular assays in zebrafish-derived cell lines

  • In vivo testing in zebrafish embryos for efficacy and toxicity

These structural studies would be particularly valuable given the ongoing interest in developing selective PGES inhibitors for potential therapeutic applications , with zebrafish models serving as an important bridge between in vitro studies and mammalian models.

What methodologies can be used to investigate the role of ptges2 in zebrafish neurodevelopment and inflammation?

Investigating the role of ptges2 in zebrafish neurodevelopment and inflammation requires multidisciplinary approaches:

• Developmental expression analysis:

  • Temporal expression profiling: qRT-PCR and Western blotting at different developmental stages

  • Spatial expression patterns: In situ hybridization and immunohistochemistry

  • Single-cell RNA-seq to identify cell-specific expression

  • Reporter transgenic lines (ptges2:GFP) to visualize dynamic expression patterns

• Loss-of-function approaches:

  • CRISPR/Cas9 knockout (as detailed in section 2.2)

  • Morpholino knockdown (for acute, early developmental studies)

  • Small molecule inhibitors of ptges2 activity

  • Photoactivatable morpholinos for temporally controlled knockdown

• Gain-of-function approaches:

  • mRNA injection for overexpression

  • Transgenic lines with inducible ptges2 expression

  • Direct microinjection of PGE2 to bypass ptges2 function

• Neurodevelopmental assessment:

  • Neuroanatomical analysis: Brain morphology, axon pathfinding, synaptogenesis

  • Functional assays: Calcium imaging, electrophysiology

  • Behavioral testing: Locomotor activity, sensory responses, learning assays

  • Transcriptomic analysis of neural tissues in wildtype vs. ptges2-deficient zebrafish

• Inflammation models:

  • Tail fin injury model

  • Lipopolysaccharide (LPS) injection

  • Pathogen infection models

  • Heat shock or chemical stress induction

• Readouts for inflammatory responses:

  • Neutrophil/macrophage recruitment using transgenic reporter lines

  • Cytokine expression profiling

  • Prostaglandin measurements in tissue extracts

  • Response to known anti-inflammatory compounds

This research direction is particularly relevant given that mammalian studies have implicated PGES enzymes in processes like fever generation, sickness behavior, inflammatory pain, and neural disease , making zebrafish an excellent model to dissect these complex physiological processes in vivo.

What are common challenges when working with recombinant Danio rerio ptges2 and how can they be addressed?

Working with recombinant Danio rerio ptges2 presents several challenges that researchers should anticipate and address:

• Protein stability issues:

  • Challenge: Protein degradation during storage or experimentation

  • Solution: Add protease inhibitors during purification, store with glycerol (5-50%), and avoid repeated freeze-thaw cycles

  • Monitor: Regular SDS-PAGE analysis of stored protein samples

• Enzymatic activity loss:

  • Challenge: Decrease in catalytic efficiency over time

  • Solution: Store in smaller aliquots, add reducing agents if appropriate, optimize buffer conditions

  • Monitor: Regular activity assays with controls

• Solubility limitations:

  • Challenge: Protein aggregation or precipitation

  • Solution: Include mild detergents or membrane mimetics if needed

  • Monitor: Dynamic light scattering or size exclusion chromatography

• Expression variability:

  • Challenge: Inconsistent protein yields between batches

  • Solution: Standardize expression protocols, monitor cell density and growth conditions

  • Monitor: Track OD600 during growth and induction

• Detection difficulties:

  • Challenge: Insufficient sensitivity in activity assays

  • Solution: Optimize antibody concentrations for Western blots, use more sensitive detection methods for PGE2 (LC-MS/MS instead of ELISA)

  • Monitor: Include positive controls in all detection methods

Methodical troubleshooting of these issues will enhance experimental reproducibility and reliability when working with this recombinant protein.

How can I design structure-function studies for Danio rerio ptges2?

Designing effective structure-function studies for Danio rerio ptges2 requires a systematic approach:

• Sequence analysis and target selection:

  • Perform multiple sequence alignment with mammalian orthologs

  • Identify conserved domains and catalytic residues

  • Target residues in these regions with point mutations:

    • Catalytic residues (particularly cysteines in the CPFCSK motif)

    • Substrate binding residues

    • Membrane interaction domains

    • Potential regulatory sites

• Mutation design strategy:

  Mutation Type	Purpose	Examples
  Conservative	Test importance of physiochemical properties	Cys→Ser, Asp→Glu
  Non-conservative	Disrupt function	Cys→Ala, Asp→Ala
  Charge reversal	Test electrostatic interactions	Asp→Lys, Lys→Glu
  Truncations	Test domain contributions	N-terminal or C-terminal deletions
  Chimeras	Domain swapping with other PGES enzymes	ptges2/cPGES hybrids

• Expression and purification:

  • Express wildtype and mutant proteins under identical conditions

  • Purify to similar levels of homogeneity

  • Verify structural integrity through circular dichroism or thermal shift assays

• Functional characterization:

  • Enzymatic activity (PGH2→PGE2 conversion rate)

  • Substrate binding affinity

  • Thermal stability

  • Oligomerization state

  • Membrane association properties

• Data analysis and interpretation:

  • Quantitative comparison of mutants to wildtype

  • Correlation of functional changes with structural predictions

  • Integration with available knowledge from mammalian orthologs

These structure-function studies will provide valuable insights into the catalytic mechanism and regulatory features of Danio rerio ptges2, which may have implications for understanding the broader PGES enzyme family.

What are the best methods for studying ptges2 interactions with other proteins in the arachidonic acid pathway?

Studying ptges2 interactions with other proteins in the arachidonic acid pathway requires multiple complementary approaches:

• In vitro protein-protein interaction studies:

  • Pull-down assays using recombinant His-tagged ptges2

  • Surface plasmon resonance (SPR) for quantitative binding kinetics

  • Isothermal titration calorimetry (ITC) for thermodynamic parameters

  • Native gel electrophoresis to detect stable complexes

  • Analytical ultracentrifugation to determine stoichiometry

• Proximity-based cellular assays:

  • Bimolecular fluorescence complementation (BiFC)

  • Förster resonance energy transfer (FRET)

  • Proximity ligation assay (PLA)

  • Cross-linking followed by immunoprecipitation

• Co-localization studies:

  • Immunofluorescence microscopy

  • Subcellular fractionation followed by Western blotting

  • Live-cell imaging with fluorescently tagged proteins

• Functional coupling experiments:

  • Coupled enzyme assays with COX enzymes

  • Reconstituted systems with multiple purified enzymes

  • Metabolic flux analysis in cells with modified ptges2 levels

• Interactome mapping:

  • Immunoprecipitation coupled with mass spectrometry

  • Yeast two-hybrid or mammalian two-hybrid screening

  • Protein microarray screening

• Key protein interactions to investigate:

  Protein	Interaction Rationale	Experimental Approach
  COX-1/COX-2	Functional coupling for PGE2 synthesis	Co-IP, coupled enzyme assays
  cPGES	Potential redundancy or compensation	Co-expression studies
  mPGES-1	Pathway coordination	Differential expression analysis
  PGE2 receptors	Signaling feedback	Receptor binding assays
  Hsp90	Chaperone interaction (known for cPGES)	Co-IP, chaperone inhibition studies

These interaction studies will help elucidate the functional integration of ptges2 within the complex prostaglandin synthesis pathway and may reveal regulatory mechanisms not yet appreciated from studies of individual enzymes.

How can Danio rerio ptges2 research contribute to understanding evolutionary conservation of eicosanoid signaling?

Studying Danio rerio ptges2 offers unique opportunities to explore the evolutionary conservation of eicosanoid signaling:

• Comparative genomics approach:

  • Phylogenetic analysis of PGES enzymes across vertebrate lineages

  • Identification of conserved regulatory elements in ptges2 gene promoters

  • Analysis of gene synteny to trace evolutionary history

  • Assessment of selection pressures (dN/dS ratios) on functional domains

• Functional conservation analysis:

  • Heterologous expression of fish ptges2 in mammalian cells

  • Complementation studies in knockout systems

  • Cross-species enzyme kinetics comparison

  • Structural comparison of substrate binding pockets

• Developmental role comparison:

  • Embryonic expression patterns across species

  • Phenotypic analysis of ptges2 knockout/knockdown in different model organisms

  • Rescue experiments with orthologs from different species

• Signaling pathway integration:

  • Comparative analysis of PGE2 receptor expression and signaling

  • Response to evolutionarily conserved stressors (infection, injury)

  • Tissue-specific functions across species

• Research implications:

  • Understanding the ancestral functions of prostaglandin signaling

  • Identifying core conserved functions versus species-specific adaptations

  • Informing translational research by highlighting fundamental signaling mechanisms

This evolutionary perspective is particularly valuable considering that prostaglandin signaling is ancient and conserved across vertebrates, yet shows important species-specific adaptations that may reflect environmental and physiological differences.

What are the emerging techniques for studying the role of ptges2 in zebrafish models of human disease?

Emerging techniques for studying ptges2 in zebrafish models of human disease combine cutting-edge molecular methods with the unique advantages of the zebrafish model:

• Advanced genetic manipulation approaches:

  • Prime editing for precise genetic modifications

  • Base editing for specific nucleotide changes

  • Inducible CRISPR systems for temporal control

  • Tissue-specific mutagenesis using Cre-lox or GAL4/UAS systems

• High-resolution imaging technologies:

  • Light sheet microscopy for whole-organism imaging

  • Super-resolution microscopy for subcellular localization

  • Intravital microscopy for real-time in vivo visualization

  • 4D imaging for developmental processes

• Single-cell and spatial transcriptomics:

  • Single-cell RNA-seq to identify cell-specific responses

  • Spatial transcriptomics to map gene expression territories

  • Trajectory inference to track developmental processes

  • Multi-omics integration (transcriptome, proteome, metabolome)

• Human disease modeling approaches:

  • Patient-derived xenograft studies

  • CRISPR knock-in of human disease mutations

  • High-throughput drug screening using disease phenotypes

  • Tissue-specific rescue with human orthologs

• Promising disease models involving ptges2:

  Disease	Zebrafish Approach	Relevance to ptges2
  Neuroinflammation	Brain-specific ptges2 manipulation	PGE2's role in neuroinflammatory processes
  Fever response	Temperature preference assays	PGES enzymes in fever generation
  Pain sensitization	Nocifensive behavior testing	PGE2's role in nociception
  Neurodegenerative disease	Transgenic neurodegeneration models	Potential protective/pathological roles
  Inflammatory conditions	Tailfin injury, chemical exposure	Prostaglandin regulation of inflammation

These advanced techniques, when applied to zebrafish models, provide powerful tools for understanding ptges2's role in human disease pathophysiology and for identifying potential therapeutic interventions.

What are the recommended controls and validation steps for ptges2 antibody-based experiments?

When conducting antibody-based experiments with Danio rerio ptges2, the following controls and validation steps are essential for reliable results:

• Antibody validation requirements:

  • Western blot verification showing a single band at expected molecular weight (~42 kDa plus tag size)

  • Loss of signal in ptges2 knockout/knockdown samples

  • Peptide competition assay showing reduced signal with blocking peptide

  • Cross-reactivity testing with related PGES family members

  • Testing in multiple applications (Western blot, IHC, IF, IP) if intended for multiple uses

• Positive and negative controls for experiments:

  • Positive controls: Tissues/cells known to express ptges2

  • Negative controls: Tissues/cells with minimal expression

  • Recombinant protein standards: Purified Danio rerio ptges2

  • Genetic controls: ptges2 knockout/knockdown samples

• Standardization procedures:

  • Antibody titration to determine optimal concentration

  • Consistent sample preparation protocols

  • Inclusion of loading controls for Western blots

  • Standardized imaging parameters for microscopy

• Cross-species reactivity considerations:

  • Test antibody specificity across species if using antibodies raised against mammalian PTGES2

  • Determine epitope conservation through sequence alignment

  • Validate in zebrafish samples even if antibody was raised against mammalian protein

• Documentation and reporting standards:

  • Record antibody source, catalog number, lot number

  • Document all experimental conditions and controls

  • Report validation data alongside experimental results

  • Share validation data with research community

Following these rigorous validation steps ensures that antibody-based experiments produce reliable and reproducible results, which is particularly important given the challenges of antibody specificity in cross-species applications.

What are the practical considerations for designing a multi-protein expression system for studying the prostaglandin synthesis pathway?

Designing a multi-protein expression system for studying the prostaglandin synthesis pathway requires careful planning:

• Expression vector design strategies:

  • Polycistronic vectors with multiple open reading frames

  • Dual/multi-promoter vectors with different strength promoters

  • Compatible vectors with different selection markers

  • Inducible expression systems for temporal control

• Protein tagging considerations:

  • Different affinity tags for each protein (His, FLAG, GST, etc.)

  • Tag position optimization (N- vs C-terminal)

  • Inclusion of protease cleavage sites

  • Fluorescent protein fusions for localization studies

• Host system selection:

  • E. coli: Simple but lacks post-translational modifications

  • Insect cells: Better for eukaryotic proteins, membrane proteins

  • Mammalian cells: Most native environment but lower yields

  • Cell-free systems: Rapid screening of conditions

• Expression optimization:

  Protein	Expression Challenges	Optimization Strategy
  COX-1/2	Membrane proteins	Detergent screening, membrane mimetics
  ptges2	Solubility issues	Fusion tags, lower temperature
  cPGES	Co-factor requirements	Glutathione supplementation
  PLA2	Potential toxicity	Tight regulation of expression

• Functional reconstitution considerations:

  • Substrate delivery mechanisms

  • Cofactor requirements

  • Membrane/detergent environment

  • Detection methods for each sequential product

• Workflow design:

  • Initial validation of individual protein expression

  • Pairwise co-expression tests

  • Sequential addition of pathway components

  • Comprehensive pathway reconstitution

This systematic approach allows researchers to rebuild the prostaglandin synthesis pathway in a controlled environment, enabling detailed mechanistic studies that would be difficult in more complex cellular systems.

What computational resources and bioinformatics tools are most valuable for ptges2 research?

The following computational resources and bioinformatics tools are particularly valuable for Danio rerio ptges2 research:

• Sequence analysis tools:

  • Ensembl Genome Browser: Gene structure and comparative genomics

  • UniProt (Entry: Q7ZUC7): Curated protein information

  • BLAST: Homology searches across species

  • Clustal Omega: Multiple sequence alignment

  • MEGA X: Phylogenetic analysis of PGES family evolution

• Structural prediction and analysis:

  • AlphaFold2/RoseTTAFold: Protein structure prediction

  • PyMOL/Chimera: Structural visualization and analysis

  • ConSurf: Evolutionary conservation mapping onto structure

  • CASTp: Binding pocket identification

  • ZDOCK: Protein-protein docking

• Functional prediction tools:

  • PROVEAN/SIFT: Predicting impact of amino acid substitutions

  • NetPhos: Phosphorylation site prediction

  • SignalP: Signal peptide prediction

  • TMHMM: Transmembrane domain prediction

  • GPS-SUMO: SUMOylation site prediction

• Zebrafish-specific resources:

  • ZFIN (Zebrafish Information Network): Comprehensive zebrafish data

  • Zebrafish Genome Browser: Genome visualization

  • ZebrafishMine: Data mining and integration

  • CRISPRscan: Guide RNA design for zebrafish

  • Zebrafish Atlas: Expression pattern database

• Literature mining and data integration:

  • PubMed/Google Scholar: Literature searches

  • Reactome: Pathway analysis

  • STRING: Protein-protein interaction networks

  • GEO/ArrayExpress: Gene expression data repositories

  • Gene Ontology: Functional annotation

These computational resources provide essential support for experimental design, data analysis, and integration of findings into the broader context of prostaglandin biology across species.